Project Summary: A coordinated and integrated response to DNA damage is critical for cells to survive and proliferate after genotoxic stress. Cells contain an elaborate molecular machinery that halts the cell cycle and repairs damaged DNA after a wide variety of extrinsic or intrinsic insults such as exposure to ultraviolet and ionizing radiation, environmental toxins, products of normal metabolism, and unstable DNA structures formed during gene transcription and DNA replication. Although DNA damage has clear potential for mutagenesis and tumor induction in normal tissues, it paradoxically forms a mainstay of modern cancer treatment due to its cytotoxic effect. Selective tumor killing by DNA damaging treatments such as radiation therapy relies on a differential response to genotoxic stress between normal and neoplastic tissue. Unfortunately, a significant number of tumor types are resistant to radiation treatment. The identification of small molecules capable of modulating the response of cells to ionizing radiation would be of tremendous utility in deciphering the molecular details of the DNA damage response, providing chemical tools for exploring the basic cell biology of environmentally-induced genetic damage, and identifying lead compounds for the development of clinically useful radiation sensitizers or radioprotective agents. In this proposal we refine, adapt and optimize an automated microscopy-based assay to simultaneously interrogate multiple components of the DNA damage response in human cancer cells for use in high- throughput screening of compounds by NIH Molecular Libraries Production Centers Network as part of the NIH Molecular Libraries and Imaging Roadmap Initiative. This assay uses novel digital image analysis techniques to create a unique dataset of multiple quantitative measurements on individual cells that optimizes the chances of success in subsequent high-throughput small molecule screening. Narrative In this proposal we refine, adapt and optimize an automated microscopy-based assay to simultaneously interrogate multiple components of the DNA damage response in human cancer cells for use in high-throughput screening of compounds by NIH Molecular Libraries Production Centers Network as part of the NIH Molecular Libraries and Imaging Roadmap Initiative. The results of this project should facilitate the development of chemical tools for studying the response of cells to environmental radiation and toxins that damage the genome, and help identify compounds that could be used as radio- protective and radio-sensitizing agents to enhance current forms of cancer treatment. [unreadable] [unreadable] [unreadable]